Versican A-subdomain is required for its adequate function in dermal development.

Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using VcanΔ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, VcanΔ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and VcanΔ3/Δ3 mice died at the neonatal period (P0). At P0, VcanΔ3/Δ3 dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in VcanΔ3/Δ3 fibroblasts as compared with that in wild type (WT). Transforming growth factor ß (TGFß) signaling mediated through the Smad2/3-dependent pathway was down-regulated in VcanΔ3/Δ3 fibroblasts and a reduced TGFß storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFß signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFß signaling.

Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains.

Hox expression in the direct-type developing sand dollar Peronella japonica.

BACKGROUND: Echinoderms are a curious group of deuterostomes that forms a clade with hemichordates but has a pentameral body plan. Hox complex plays a pivotal role in axial patterning in bilaterians and often occurs in a cluster on the chromosome. In contrast to hemichordates with an organized Hox cluster, the sea urchin Strongylocentrotus purpuratus has a Hox cluster with an atypical organization. However, the current data on hox expression in sea urchin rudiments are fragmentary. RESULTS: We report a comprehensive examination of hox expression in a sand dollar echinoid. Nine hox genes are expressed in the adult rudiment, which are classified into two groups, but hox11/13b belongs to both: one with linear expression in the coelomic mesoderm and another with radial expression around the adult mouth. The linear genes may endow the coelom/mesentery with axial information to direct postmetamorphic transformation of the digestive tract, whereas the radial genes developmentally correlate with the morphological novelties of echinoderms and/or sea urchins. Recruitment of the radial genes except hox11/13b appears to be accompanied by the loss of ancestral/axial roles. CONCLUSIONS: This in toto co-option of the hox genes provides insight into the molecular mechanisms underlying the evolution of echinoderms from a bilateral ancestor.

Chondroitin sulfate-liposome: clustering toward high functional efficiency.

Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids, and generated CSA- and CSE-liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE-liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS-liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.

Unusual coelom formation in the direct-type developing sand dollar Peronella japonica.

Peronella japonica is a sand dollar with a zygote that develops into an abbreviated pluteus but then metamorphoses on day three. The adult rudiment formation is unique; it uses a median position of the hydrocoel and a stomodeum-like invagination of vestibule that covers the dorsal side of the hydrocoel. However, the developmental processes underlying coelom formation remain unclear. In this study, we examined this process by reconstructing three-dimensional images from serial sections of larvae. We show that the left coelom developed by both schizocoely and enterocoely from the archenteron tip, whereas the hydrocoel and right coelom formed by enterocoely from the archenteron. This coelom formation arranged the coelomic compartments directly along the adult oral-aboral axis by skipping the initial bilateral phases. Furthermore, our data indicate P. japonica retains ancestral asymmetry along the left-right axis in the location of the adult rudiment.

Krüppel-like is required for nonskeletogenic mesoderm specification in the sea urchin embryo.

The canonical Wnt pathway plays a central role in specifying vegetal cell fate in sea urchin embryos. SpKrl has been cloned as a direct target of nuclear beta-catenin. Using Hemicentrotus pulcherrimus embryos, here we show that HpKrl controls the specification of secondary mesenchyme cells (SMCs) through both cell-autonomous and non-autonomous means. Like SpKrl, HpKrl was activated in both micromere and macromere progenies. To examine the functions of HpKrl in each blastomere, we constructed chimeric embryos composed of blastomeres from control and morpholino-mediated HpKrl-knockdown embryos and analyzed the phenotypes of the chimeras. Micromere-swapping experiments showed that HpKrl is not involved in micromere specification, while micromere-deprivation assays indicated that macromeres require HpKrl for cell-autonomous specification. Transplantation of normal micromeres into a micromere-less host with morpholino revealed that macromeres are able to receive at least some micromere signals regardless of HpKrl function. From these observations, we propose that two distinct pathways of endomesoderm formation exist in macromeres, a Krl-dependent pathway and a Krl-independent pathway. The Krl-independent pathway may correspond to the Delta/Notch signaling pathway via GataE and Gcm. We suggest that Krl may be a downstream component of nuclear beta-catenin required by macromeres for formation of more vegetal tissues, not as a member of the Delta/Notch pathway, but as a parallel effector of the signaling (Krl-dependent pathway).

The Hox8 of the hemichordate Balanoglossus misakiensis.

Deuterostomes comprise a monophyletic group of animals that include chordates, xenoturbellids, and the Ambulacraria, which consists of echinoderms and hemichordates. The ancestral chordate probably had 14 Hox genes aligned linearly along the chromosome, with the posterior six genes showing an independent duplication compared to protostomes. In contrast, ambulacrarians are characterized by a duplication of the posterior Hox genes, resulting in three genes known as Hox11/13a, Hox11/13b, and Hox11/13c. Here, we isolated 12 Hox genes from the hemichordate Balanoglossus misakiensis and found an extra Hox gene that has not been reported in hemichordates. The extra B. misakiensis gene was suggested to be Hox8 from paralog-characteristic residues in its hexapepetide motif and homeodomain and a comparison with Strongylocentrotus purpuratus Hox genes. Our data suggest that the ancestor of echinoderms and hemichordates may have had a full complement of 12 Hox genes.

Interaction of receptor type of protein tyrosine phosphatase sigma (RPTPσ) with a glycosaminoglycan library.

Receptor type of protein tyrosine phosphatase sigma (RPTPσ) functions as a glycosaminoglycan (GAG) receptor of neuronal cells in both the central and peripheral nervous systems. Both chondroitin sulphate (CS) and heparan sulphate (HS) are important constituents of GAG ligands for RPTPσ, although they have opposite effects on neuronal cells. CS inhibits neurite outgrowth and neural regeneration through RPTPσ, whereas HS enhances them. We prepared recombinant RPTPσ N-terminal fragment containing the GAG binding site and various types of biotin-conjugated GAG (CS and HS) with chemical modification and chemo-enzymatic synthesis. Then interaction of the RPTPσ N-terminal fragment was analysed using GAG-biotin immobilized on streptavidin sensor chips by surface plasmon resonance. Interaction of RPTPσ with the CS library was highly correlated to the degree of disulphated disaccharide E unit, which had two sulphate groups at C-4 and C-6 positions of the N-acetylgalactosamine residue (CSE). The optimum molecular mass of CSE was suggested to be approximately 10 kDa. Heparin showed higher affinity to RPTPσ than the CS library. Our GAG library will not only contribute to the fields of carbohydrate science and cell biology, but also provide medical application to regulate neural regeneration.

Experimental Approach Reveals the Role of alx1 in the Evolution of the Echinoderm Larval Skeleton.

Over the course of evolution, the acquisition of novel structures has ultimately led to wide variation in morphology among extant multicellular organisms. Thus, the origins of genetic systems for new morphological structures are a subject of great interest in evolutionary biology. The larval skeleton is a novel structure acquired in some echinoderm lineages via the activation of the adult skeletogenic machinery. Previously, VEGF signaling was suggested to have played an important role in the acquisition of the larval skeleton. In the present study, we compared expression patterns of Alx genes among echinoderm classes to further explore the factors involved in the acquisition of a larval skeleton. We found that the alx1 gene, originally described as crucial for sea urchin skeletogenesis, may have also played an essential role in the evolution of the larval skeleton. Unlike those echinoderms that have a larval skeleton, we found that alx1 of starfish was barely expressed in early larvae that have no skeleton. When alx1 overexpression was induced via injection of alx1 mRNA into starfish eggs, the expression patterns of certain genes, including those possibly involved in skeletogenesis, were altered. This suggested that a portion of the skeletogenic program was induced solely by alx1. However, we observed no obvious external phenotype or skeleton. We concluded that alx1 was necessary but not sufficient for the acquisition of the larval skeleton, which, in fact, requires several genetic events. Based on these results, we discuss how the larval expression of alx1 contributed to the acquisition of the larval skeleton in the putative ancestral lineage of echinoderms.